In most cell types, microtubules are organized by the centrosome, a specialized organelle composed of an orthogonal pair of centrioles surrounded by a matrix of pericentriolar material (PCM). During the cell cycle, the centrosome duplicates precisely once. This event is of critical importance to mitotic spindle assembly as it ensures that two centrosomes are available to form the poles of the bipolar spindle. Duplication involves splitting of the existing centriole pair followed by the synthesis of a new centriole next to each old centriole. As the cell progresses toward mitosis, the centrosome matures as it gradually acquires increased levels of microtubule nucleating capacity and PCM. Despite the importance of centrosome duplication and maturation, little is known of how these processes are regulated at a molecular level. In my laboratory, we are using the nematode Caenorhabditis elegans to study centrosome duplication and maturation. Specifically, our goals are to identify the factors that regulate these processes and to understand how they function on a molecular level. [unreadable] During 2007, considerable effort was applied to the study of two factors that regulate centrosome duplication. Both were identified in a genetic screen for suppressors of a zyg-1 reduction-of-function mutation. SZY-20 is a negative regulator of ZYG-1 that localizes to the nucleus, nucleolus and centrosomes. Our analysis indicates that SZY-20 limits centrosome size. In szy-20 mutant embryos, centrosomes are enlarged and possess elevated levels of ZYG-1 and PCM components such as SPD-2 and SPD-5. Such embryos however do not over-express these proteins, as overall levels of ZYG-1, SPD-2 and SPD-5 are similar to wild-type embryos. We have found that these enlarged centrosomes nucleate more microtubules than their wild-type counterparts and that the enlarged centrosome phenotype is associated with defects in a number of microtubule-dependent processes. Of particular significance we find that ZYG-1 plays a critical role in defining centrosome size; inhibition of ZYG-1 in the szy-20 mutant restores normal centrosome size and rescues the microtubule-related defects. Our work has thus identified a novel ZYG-1-dependent cellular mechanism that regulates centrosome size and microtubule-nucleating capacity.[unreadable] We have cloned the szy-20 gene and find that it encodes a widely conserved, yet unstudied protein. Through protein database searches and sequence analysis we determined that SZY-20 belongs to a conserved family of likely RNA-binding proteins. We have now confirmed this by demonstrating that SZY-20 has RNA-binding activity in vitro. All together, our data indicates that SZY-20 acts locally to regulate the association of ZYG-1 with the centrosome. SZY-20 might achieve this by regulating expression of centrosome-associated mRNAs or through protein-protein interactions with centrosome components. [unreadable] We have also been studying another negative regulator of ZYG-1 named SZY-5. Unlike SZY-20, SZY-5 does not seem to regulate centrosome-levels of ZYG-1 and thus appears to interact with ZYG-1 in a manner distinctly different from that of SZY-20. In support of this we find that loss of szy-5 activity produces a cytological phenotype distinct from that of szy-20 mutants. In szy-5 mutants tubulin assembles into large cytoplasmic aggregates in both embryos and germ cells. Interestingly, centriolar proteins co-localize with these aggregates, suggesting they represent abnormal intermediates in the centrosome duplication pathway. We have further found that this aggregation phenotype is sex specific; that is, aggregates only form in female germ cells but not male. This might be due to the high concentrations of centriole proteins that are produced in the female germ line and stored in the oocyte for use in the embryonic cell divisions. We have cloned szy-5 and find it encodes a protein with four zinc fingers and a coiled-coil domain. Currently we are working to determine where in the cell SZY-5 localizes and have prepared polyclonal rabbit sera against SZY-5. Our preliminary analysis indicates the protein might localize to centrosomes, spindles, and/or nuclei.